Liquid phase and solid phase immunoassays and their numerous modifications and variations have been described in the literature for the measurement of a host of circulating antigens and antibodies. Numerous U.S. and Foreign patents have been issued dealing with one or more aspects of immunoassay techniques.
Competitive isotopic double antibody separation immunoassay techniques have played a substantial role in the measurement of antigens and haptens. Such methods are sensitive and reproducible but require centrifugation to separate the free labeled antigen or hapten from the antibody bound label. Although such methods utilize first order liquid phase kinetics, they rely on polyethylene glycol catalyzed second antibody separation. Several drawbacks are inherent in such systems; namely, (a) the stringent requirement of a highly purified antigen or hapten for isotopic labeling and (b) high nonspecific binding due in part to tracer impurity and instability and/or polyethylene glycol-second antibody separation.
Competitive enzyme double antibody separation immunoassay techniques have been used on a much smaller scale. Antigen or hapten enzyme labels, although more stable than their isotopic counterparts, suffer in such assays due to extremely high nonspecific bindings and require multiple washings thus reducing sensitivity and reproducibility.
Substituting fluorescent or chemiluminescent labels in said double antibody competitive type assays have not fully resolved the intrinsic problem of non-specific binding and limited sensitivity and reproducibility of such methods. Homogeneous enzyme immunoassays as described in U.S. Pat. No. 3,817,837 have in part addressed the non-specific binding problem described above for non-isotopic competitive double antibody separation techniques. However, the teachings of said U.S. Pat. No. 3,817,837 are limited to small molecular weight haptens that are present in fairly high concentrations in body fluids.
Fluorescent polarization competitive immunoassay as described in U.S. Pat. No. 4,420,568 has successfully eliminated most of the sensitivity problems described in U.S. Pat. No. 3,817,837 but so far their applications have been limited to small molecules. Other competitive fluorescent immunoassays have since been reported for large molecular weight antigens but like fluorescent polarization require a special dedicated instrument for their practice.
Competitive solid phase immunoassays have been in use for the past decade and have gradually replaced competitive double antibody separation techniques for small molecular weight haptens and several larger antigens with a molecular mass of less than 30,000 daltons. Such isotopic competitive solid phase immunoassays are virtually free of non-specific binding problems associated with liquid phase competitive immunoassays but are not applicable to large molecular weight antigens due mainly in part to steric hindrance on the solid support.
Solid phase non-isotopic competitive immunoassays suffer from similar problems of steric hindrance on the solid support and in case of enzyme labels the size of the label itself adds to steric problem as well. The present invention will address some of the steric hindrance problems of solid phase supports inherent in competitive solid phase assays. Solid phase immunometric isotopic and non-isotopic assays have been well developed for the measurement of large molecular mass polyvalent antigens and antibodies. Several methods have been published and lately several U.S. and Foreign patents have been issued describing different aspects of this analytical technique.
U.S. Pat. No. 3,654,090 was one of the earliest teachings of a sequential two-step immuno-enzymometric assay for the detection of polyvalent antigens. Several modifications of the original teachings of said patent have since been applied to the measurement of high molecular mass antigens using either isotropic or non-isotopic signal producing probes.
For obvious analytical reasons it is well established that the use of two different polyclonal antibodies raised in different species directed at the same antigen (one immobilized on solid support and the second labeled with a signal producing probe) enhances the sensitivity of the assay and reduces to a certain extent background signals in immunometric assays. U.S. Pat. No. 4,376,110 teaches the use of two monoclonal antibodies directed at two different epitopes on polyvalent antigens in immunometric assays. Unlike U.S. Pat. No. 3,654,090, U.S. Pat. No. 4,376,110 uses a co-incubation non-sequential assay system. Similarly, U.S. Pat. No. 4,474,892 also describes a two-site immunometric assay system using monoclonal antibodies of different classes or subclasses directed at the same antigen. The above-described methods although they achieve acceptable sensitivity, specificity and to a certain extend reduced background signals, all suffer from low reaction kinetics due to the immobilization of the antibody on solid support and a solid phase type reaction. It is well established that solid phase reactions have lower reaction kinetics than liquid phase reactions and also have lower signal to background ratio even if the immobilized antibody has a higher affinity constant prior to immobilization. This is largely due to steric hindrance effects on the solid support. If such methods are not properly optimized in so far as the quantity of immobilized antibody and the concentration and specific activity of the probe producing signal attached to the second antibody are concerned, the so-called "high-dose hook effect" could jeopardize the reliability and validity of such methods.
A further disadvantage of immunometric methods is the inconsistency of the antibody immobolizing process from batch to batch. U.S. Pat. No. 4,496,654 discloses this problem of inconsistent immobilization of antibodies and teaches that by first immobilizing avidin on a solid support then reacting said support with biotinylated antibody to form a solid phase antibody support, uniform immobilization is achieved. However, in this disclosed modification slower reaction kinetics are inevitable and the dangers of a "high-dose hook effect" are still probable. The present invention addresses these shortcomings and the disadvantages inherent in these immunometric assays.
Modifications of classical immunometric assays for the detection of antigens has also been extended for the measurement of specific antibodies by using immobilized antigens as the immunosorbant. A notable application of such techniques has been widely used for the detection of allergen specific immunoglobulins, IgE (reagin-immunoglobulins). Historically, specific allergen testing has followed the teachings of U.S. Pat. No. 3,720,760 and its Foreign counterparts, in which specific allergens are immobilized on a solid support (mainly filter paper discs) and reacted with a patient sample suspect of containing the allergen specific immunoglobulin IgE. After an initial incubation (typically 24 hours) the solid support is washed to remove any non-specific binding from the serum components, then the solid support is allowed to react with a labeled anti-IgE antibody. After a second wash step of the solid support it is checked for the presence of labeled material. This approach, although widely used has several shortcomings; namely, slow reaction kinetics due to the solid phase reactions, difficulty of producing immobilized allergens that have the same binding characteristics as the natural allergens for IgE and the inconsistency of producing a solid phase allergen from batch to batch.
Recently, Aalberse, et al. (J. Imm. Methods 87: 51-57 (1986)) decribe the use of hapten-modified antigens instead of solid phase coupled antigens in a radioallergosorbent test-type assay. In such assay a patient sample suspected of containing specific allergen IgE is reacted with trinitrobenzene sulfonic acid (TNP) modified specific allergen for two hours then further reacted overnight with a solid phase coupled anti-TNP to form IgE-allergen-TNP-anti-TNP complex. The solid phase is washed and again reacted overnight with 125-I-anti-IgE antibody, rewashed and counted for the presence of 125-I isotope which is directly proportional to the concentration of allergen specific IgE in the patient sample. In this approach the authors claim to have gained the benefit of liquid phase kinetics in their first reaction but fail to substantiate the extent of TNP labeling of their allergens. Directly labeling allergens with haptens such as TNP poses the problem of missing certain vital allergenic components that might not be labeled during such a process and thus will not be quantified in said process. Also, the authors failed to show enhanced reaction timing (two days) as compared to the method described in U.S. Pat. No. 3,720,760.
The present invention circumvents the problems associated with the traditional method of allergen testing and the modifications thereof.